It has been becoming increasingly evident that in utero exposure of mammalian fetuses to environmental endocrine disruptors may cause transgenerational diseases through disruption of the epigenetic mechanisms regulating gene expression. The disease phenotypes of the F2 and later generations may resemble each other because such phenotypes might be derived from a common set of germline-transmitted epimutations. In contrast, the F1 phenotype is not derived from the germline epimutations; therefore, it does not need to be similar to phenotypes of the F2+ generations. However, many animal studies have reported remarkably similar phenotypes of the F1 and the F2+ generations. To explain this somewhat paradoxical phenomenon, we hypothesize that the nuclear receptors expressed in both the somatic cells and the germline cells may be responsible for the phenotypes commonly observed in the F1 and the F2+ generations but through distinct mechanisms. Specifically, while nuclear receptors expressed in the somatic cells may directly affect transcription of the hormone target genes to provoke the F1 phenotypes, in the germline cells the same nuclear receptors might cause epimutations at the same set of genes to cause the F2+ phenotypes. To obtain experimental evidence supporting this hypothesis, our Specific Aim 1 aims to determine whether fetal exposure of mice to endocrine disruptors known to cause transgenerational obesity introduces specific epigenetic changes at the binding sequences of the affected nuclear receptors in the genome of the germline cells. Pregnant female transgenic mice engineered for germline-specific GFP expression will be exposed to tributyltin, rosiglitazone, or Bisphenol A from 12.5 dpc until 18.5 dpc, and the GFP+ germline cells will be collected from their fetuses by FACS. Genome-wide profiling of mRNA expression, DNA methylation, and nuclear hormone receptor binding in the genome of the F1 germline cells will be determined by deep sequencing (RNA- seq, Bisulfite-seq, and ChIP-seq). To identify the transgenerationally transmittable germline epimutations, we will also determine transcriptomes and epigenetic mark distributions in the GFP+ germline cells of F2 and F3 fetuses at 18.5 dpc. To evaluate the transgenerational obesity phenotype and the germline-conveyed epimutations, we will examine fat depot weight, adipocyte size, and adipocyte number of brown and white adipose tissues in 8-weeks-old F1, F2, and F3 pups. Selected adipose tissue specimens will also be subjected to transcriptomal and epigenomic analyses. Our Specific Aim 2 aims to determine whether exposure of mouse and human primordial germ cell-like cells (PGC-LCs) to the obesogenic endocrine disruptors causes specific epigenetic changes at the nuclear receptor binding sequences to affect cellular sensitivity to the relevant hormonal agents in vitro. We will determine transcriptomes, epigenomic mark distributions, and genome-wide ligand sensitivity profiles of the hormone responsive genes using deep sequencing. RNAi knockdown and CRISPR/Cas9 gene knockout experiments will establish the requirement of each nuclear receptor and the Tet family DNA hydroxymethyltransferases in formation of the exposure-induced epimutations. Successful completion of this project will establish a novel theoretical and methodological foundation, including in vivo and in vitro model systems, for future research on the molecular mechanisms of transgenerational diseases caused by fetal exposure to endocrine disruptors.